Toxin production is essential for the virulence of Vibrio cholerae and the pathogenesis of cholera disease. Epidemiological data indicate an increased prevalence of pathogenic strains defective in the production of cholera toxin. Reactogenic symptoms are attributed to a number of accessory toxins, including the V. cholerae RTX toxin. However, unlike the other members of the RTX family, RtxA is not a pore-forming toxin. RtxA activity promotes host cell rounding, and actin monomers are covalently cross-linked through a completely novel mechanism. Understanding the individual components of the cross-linking reaction is the focus of this proposal. Cytoskeletal inhibition will define the actin substrate, the characterization of a putative enzymatic domain may identify a catalytic function of RtxA, and a combined biochemical and genetic approach will reveal the chemical properties of the linkage between actin monomers.